Background

Tissue Collection

Western blotting can be used for protein detection in a variety of sample types (e.g. blood), but typically brain tissue is used in neuroscience applications. Fresh brain tissue should be snap-frozen either via isopentane on dry ice or liquid nitrogen. Perfused brain tissue cannot be used for western blotting because perfusion ‘fixes’ proteins in their quatrinary formations, meaning that they cannot be denatured.

Dissection

The process that you will follow to dissect region(s) of interest will depend on your specific experimental parameters

A “graphical abstract” of the Western Blotting Process:

(borrowed from the Wikipedia page on Western Blotting, which has tons of useful info):

Tissue Processing

Purpose: to break cells open so that all the proteins inside them are “dumped” into a “protein soup”

Basic tissue processing for whole-cell lysates:

Use a more refined tissue processing protocol to separate out sub-cellular compartments

Bradford Assay

Purpose: To measure protein concentrations in each sample

Materials

Procedure

Prepare Standard Curve:

  • Label a set of tubes A:E
  • Add 500 ul RO to each
  • Thaw an aliquot of 1 mg/ml BSA (-20 freezer, bottom shelf, left side)
  • Add 500 ul STOCK BSA to tube A
  • Serial dilute down the line by half:
Label Protein Concentration RO to Add BSA to Add
Blank 0 500 Nothing
A 1 500 500 STOCK
B 0.5 500 500 A
C 0.25 500 500 B
D 0.125 500 500 C
E 0.0625 500 500D

Prepare Bradford Samples:

  • Label a set of tubes with sample IDs (These will be garbage after the Bradford is done)
  • add 45ul RO to each tube
  • add 5ul protien lysate -> Vortex.
    • The Bradford samples can sit at on the bench at room temperature.
    • Leave the remaining protein stocks on ice while running the Bradford assay.

Prepare the Assay Plate:

  • Set up Assay Plate (Pipette all in triplicates in a 96 well plate. Label the lid with subject numbers to make sure you know who’s who..)
    • Blank (10 µl RO)
    • Standard (10 µl A – E)
    • Samples - 10 µl each
    • Run samples in triplicate because the Bradford is a crude assay (allows you to drop one point if it’s a clear outlier from the other two - e.g. D7 in the image below).
  • Add 200ul Bradford dye to each well using the repeater pipette.
  • let stand 5-10 minutes (depends who you ask)
  • Read at 595 nm in the bio shared space (third floor)
    • The computer is slow, best to set up ahead of time.
    • Use Gen5 - same as the cytation.

Match Protein Concentrations

Purpose: To match protein concentrations in the simplest and most consistent way possible.

Dilute across two steps:

  1. Dilute with lysis buffer to 1.33 ug / ul
  2. Add 1:3 Pre-fab loading buffer** to each sample

** Add BME as per the label instructions to a tube of loading dye right before use. Don’t add BME to the stock bottle - it isn’t shelf stable

Western Blots: Day 1 (of 2)

Overview of steps to run the western gel:

Step #1: Casting Gels

Materials

  • BioRad FastCast Gel kit
    • The gel percentage can be adjusted based on the size of your protein of interest:
  • Temed (4 degrees - common chemicals tray)
  • 10% APS (4 degrees with the Temed OR aliquated @ -20 - bottom shelf left side)
    • To make stock: 1 gram APS powder + 10 ml RO -> dissolve and aliquot in 1ml tubes.
  • 15 mL falcon tubes (2 - label one “resolver” and the other “stacker”)

Procedure

  • Mix A & B for the resolver and stacker in their respective tubes
  • Add temed + APS to resolver tube -> use a 10 ml sterological pipette to pour ~4/5ths of the way up the plate.
  • Add temed + APS to the stacker tube -> use a 1ml pipette to fill the glass plates with the stacker solution until it spills over the top.
  • Insert the green comb to create the wells.
    • Any chips in the glass plates will increase likelihood of the gel partially leaking out / falling / generally not working.
    • We almost always need more short glass plates because they are thin and prone to chipping.
    • Try not to cause bubbles… Bubbles are the worst.
    • If you see a bubble early, it will only grow throughout the polymerization process..
    • Can make extra gels. Unused gels can be wrapped in wet paper towels and stored in a ziploc bag @ 4 degrees.

Step #2: Prepare Run Apparatus

  • Add two plates into the apparatus with the prongs
    • The apparatus without the prongs is for running 3+ gels in the same tank (we don’t like to do that - they don’t run as nice.)
  • See this youtube video for instructions on preparing the gel apparatus and loading samples.

Step #3: Loading Samples

  • If you matched all your samples to 1 ug / ul (see “Matching Protein Concentrations”, above), load equal volumes into each lane of the gel.
    • Best practice! - yeilds the most consistent run.
  • Try not to poke the gel at all with the pipette tip.

Step #4: Run the Gel

  • Make sure that the BLACK mark on the side of the gel holding apparatus lines up with the BLACK mark on the side of the tank (and that on the other side, RED goes with RED..).
  • Ensure that the cables are attached such that BLACK-BLACK and RED-RED.
  • Failure to match the colours will result in terrible things.
  • Watch the purple line move through the gel.

also see this informative video for more info

At the end of the transfer:
  • Open the sandwiches
    • you should see the coloured bands of the ladder transferred over to the membrane.
    • throw the gel out now
    • If running multiple membranes, make them clearly identifiable in some way. (e.g., cut notches in opposite corners)
  • Mix up some “wash buffer”
    • 999ml 1x TBS
    • 1ml tween80 (pipette slowly - it’s very thick.)

Block the Membrane

Purpose: to reduce non-specific signal by blocking binding sites on the PVDF membrane to prevent primary antibody binding.

Procedure:

Primary Antiboy Incubation

Purpose: To facilitate specific binding of the primary antibody to the target antigen on the membrane.

Western Blots day 2: Secondary Antibody & Imaging

Purpose: To bind a fluorescent secondary antibody to the primary antibodies (which are bound to their target antigens) - Secondary antibody binding will facilitate detection in a fluorescent imager (e.g. the iBright 1500).

IMAGE - Save pictures on USB

You must have both a target protein and a normalizer protein (loading control) for each membrane

  • In the example below, the intensity of the GR band would be divided by the intensity of the corresponding gapdh band.